Pigment Mutants Research Articles

Galanin Signaling in the Brain Regulates Color Pattern Formation in Zebrafish.

SummaryColor patterns are prominent features of many animals and are of high evolutionary relevance. In basal vertebrates, color patterns are composed of specialized pigment cells that arrange in multilayered mosaics in the skin. Zebrafish (Danio rerio), the preeminent model system for vertebrate color pattern formation, allows genetic screens as powerful approaches to identify novel functions in a complex biological system. Adult zebrafish display a series of blue and golden horizontal stripes, composed of black melanophores, silvery or blue iridophores, and yellow xanthophores. This stereotyped pattern is generated by self-organization involving direct cell contacts between all three types of pigment cells mediated by integral membrane proteins [1, 2, 3, 4, 5]. Here, we show that neuropeptide signaling impairs the striped pattern in a global manner. Mutations in the genes coding either for galanin receptor 1A (npm/galr1A) or for its ligand galanin (galn) result in fewer stripes, a pale appearance, and the mixing of cell types, thus resembling mutants with thyroid hypertrophy [6]. Zebrafish chimeras obtained by transplantations of npm/galr1A mutant blastula cells indicate that mutant pigment cells of all three types can contribute to a normal striped pattern in the appropriate host. However, loss of galr1A expression in a specific region of the brain is sufficient to cause the mutant phenotype in an otherwise wild-type fish. Increased thyroid hormone levels in mutant fish suggest that galanin signaling through Galr1A in the pituitary is an upstream regulator of the thyroid hormone pathway, which in turn promotes precise interactions of pigment cells during color pattern formation.

Effects of body pigmentation mutations on Drosophila melanogaster mating behavior

The model of congenic strains of Drosophila melanogaster was used to investigate the peculiarities of the effect of mutations in yellow (y), ebony (e), and black (b) genes involved in biogenesis of cuticle pigments on imago mating behavior indicators. The aim of this study was to find out if the effect of the given mutations on Drosophila imago mating behavior depends on the general genetic background on which they are realized. To achieve this goal, pairs of congenic strains were constructed using successive saturation crosses followed by selection for the marker phenotype resulted in each of the mutant alleles introduced in homozygous condition into the genotype of either Canton-S or Oregon-R wild-type stock instead of the corresponding wild-type allele present in these stocks initially. Individuals of strains resulted were tested for mating receptivity of females and mating activity of males. Each of the indicators was evaluated as a proportion of sexually mature but virgin individuals of a particular sex copulated successfully within the first hour after placing them in a test chamber with an excess of individuals of the opposite sex. According to the data obtained and the results of their statistical analysis, it was proved that the introduction of a mutation into the genetic background of the wild-type stock is accompanied with a change in the studied characteristics of imago mating behavior. The effect depends on the mutation introduced and on the genotype of the recipient stock. Thus, males of the yC-S strain are characterized by increased mating activity comparatively to males of the wild-type Canton-S stock. These results expand the known effects of yellow mutation. Males of the bC-S and eC-S strains, on the contrary, are less active than the males of the wild-type Canton-S stock. The most pronounced effects on mating receptivity of females were fixed for b (an increase in the indicator when introduced into Oregon genetic background) and e (a decrease when introduced into Canton-S genetic background) mutations. The indicators studied under the conditions of the given experimental scheme change in direct proportion (rS = 0,76; p < 0,05). In other words, if the strain is characterized by high mating activity of males, as a rule, a high mating receptivity of females will be also observed.

Diversity of genetic lesions characterizes new Arabidopsis flavonoid pigment mutant alleles from T-DNA collections

The visual phenotypes afforded by flavonoid pigments have provided invaluable tools for modern genetics. Many Arabidopsis transparent testa (tt) mutants lacking the characteristic proanthocyanidin (PA) seed coat pigmentation and often failing to accumulate anthocyanins in vegetative tissues have been characterized. These mutants have significantly contributed to our understanding of flavonoid biosynthesis, regulation, and transport. A comprehensive screening for tt mutants in available large T-DNA collection lines resulted in the identification of 16 independent lines lacking PAs and anthocyanins, or with seed coat pigmentation clearly distinct from wild type. Segregation analyses and the characterization of second alleles in the genes disrupted by the indexed T-DNA insertions demonstrated that all the lines contained at least one additional mutation responsible for the tt phenotypes. Using a combination of RNA-Seq and whole genome re-sequencing and confirmed through complementation, we show here that these mutations correspond to novel alleles of ttg1 (two alleles), tt3 (two alleles), tt5 (two alleles), ban (two alleles), tt1 (two alleles), and tt8 (six alleles), which harbored additional T-DNA insertions, indels, missense mutations, and large genomic deletion. Several of the identified alleles offer interesting perspectives on flavonoid biosynthesis and regulation.

Characterization of a repertoire of tomato fruit genetic variants in the San marzano genetic background

San Marzano (SM) is a worldwide famous tomato Italian traditional landrace characterized by elongated fruits with a dual-purpose use in the fresh and processing market. A repertoire of mutations affecting the fruit and of interest for commercial breeding were introduced into the SM genetic background following backcross schemes. The lines generated included 13 genotypes each carrying a single mutation in genes controlling a) the content of all pigments (hp-1, hp-2, pd), b) of carotenoids (r, t, at, B, B_moB), c) of chlorophyll (gf), d) of flavonoids (y) or e) the ripening process (Nr, rin, Gr). Five lines carrying a combination of two mutations were also included. Analysis of SNP polymorphisms showed that the genetic distance of the lines from the recurrent parent was very variable and not well predicted by the number of backcrosses because it was also a function of the dissimilarity of the donor parent. All the genotypes, together with an SM control, were grown in two consecutive years and characterized for vegetative, reproductive and fruit quality traits. Overall, the studied lines reproduced the SM typical phenotypes, but several differences also emerged as both possible negative or advantageous pleiotropic traits for fresh or processing uses and peeling. High pigment mutations confirmed the negative pleiotropic effects on plant fertility and fruit development described earlier and also negatively affected fruit post-harvest life. These latter defects were also reported in the carotenoid mutant tangerine. In contrast, absence of peel pigmentation in the y mutant was associated with positive postharvest properties as those fruit presented higher resistance to wrinkling and dehydration. Delayed ripening mutants showed positive post-harvest phenotypes, as expected. In conclusion, the study of the present repertoire of fruit variations in an elongated tomato genotype represents a contribution to expand the study of fruit physiology to unusual fruit types and to breed innovative tomato lines with valuable nutritional and technological properties.

Characterization of an Unusually Aggressive Ovarian Malignant Sex Cord Stromal Tumor in an Adolescent Female With Peutz-Jeghers Syndrome

Abstract Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant disorder with mutations in SKT11 (19p13.3), mucocutaneous hyperpigmentation, gastrointestinal hamartomatous polyposis, and increased cancer predisposition. A PJS-associated distinctive ovarian tumor is sex cord tumor with annular tubules (SCTAT), which is multifocal, microscopic, bilateral, and almost always clinically benign. Non-SCTAT ovarian tumors with malignant behavior are exceptionally rare. We describe a malignant, multiply recurrent, widely metastatic sex cord stromal tumor in PJS. Case Report: A 17-year-old female presented with new complaints of unintentional weight loss, alopecia, and abdominal fullness. She had a PJS diagnosis at age 7 with mucocutaneous pigmentation, gastrointestinal polyposis, and STK11 mutation. CT showed a 23-cm abdominopelvic mass with diffuse peritoneal metastases and malignant pleural effusion. CA-125 was elevated. CEA, CA 19-9, β-hCG, AFP, and LDH were normal. Laparoscopic biopsy pathology revealed a tumor composed of sheets and loose aggregates of mitotic spindled and round heterogenous eosinophilic and basophilic cells within fibromyxoid stroma. Rare tubules with eosinophilic cytoplasm and focal foamy cells were also seen. IHC was significant for inhibin, calretinin, CD56 and WT1 positivity, and cytokeratin and EMA negativity, consistent with poorly differentiated sex cord stromal tumor. Interestingly, EM showed neurosecretory dense core granules and neurite-like processes. After neoadjuvant therapy, the resected left adnexal mass was 25.5 cm, purple-gray/tan-red, heterogeneous, diffusely cystic, diffusely edematous, rubbery to spongy. In the left ovary were also found unassociated microscopic SCTAT foci. During adjuvant chemotherapy, the patient became pregnant. During the pregnancy, the patient developed liver and pelvic peritoneal recurrence and continued altered chemotherapy. She delivered a healthy late-preterm infant but also concurrently had diffuse peritoneal recurrence. She underwent two more cycles of adjuvant chemotherapy. Approximately 2 years after finishing adjuvant chemotherapy, she again developed peritoneal recurrences, which were resected, and partial chemotherapy response was seen on pathology. She is currently undergoing additional chemotherapy cycles 4 years since the initial diagnosis.

The silkworm (Bombyx mori) neuropeptide orcokinin is involved in the regulation of pigmentation

The natural colorful cuticles of insects play important roles in many physiological processes. Pigmentation is a physiological process with a complex regulatory network whose regulatory mechanism remains unclear. Bombyx mori pigmentation mutants are ideal materials for research on pigmentation mechanisms. The purple quail-like (q-lp) and brown quail-like (q-lb) mutants originated from plain silkworm breeds 932VR and 0223JH respectively exhibit similar cuticle pigmentation to that of the quail mutant. The q-lp mutant also presents a developmental abnormality. In this study, genes controlling q-lp and q-lb mutants were located on chromosome 8 by positional cloning. Then the neuropeptide gene orcokinin (OK) was identified to be the major gene responsible for two quail-like mutants. The B. mori orcokinin gene (BommoOK) produces two transcripts, BommoOKA and BommoOKB, by alternative splicing. The CRISPR/Cas9 system and orcokinin peptides injection were used for further functional verification. We show a novel function of BommoOKA in inhibiting pigmentation, and one mature peptide of orcokinin A, OKA_type2, is the key factor in pigmentation inhibition. These results provide a reference for studying the function of orcokinin and are of theoretical importance for studying the regulatory mechanism of pigmentation.

Genetic analysis of the period of meiosis and morphogenesis of the gametophytic blades in Pyropia suborbiculata (Bangiales, Rhodophyta)

A crossing experiment between two artificial pigmentation mutants of Pyropia suborbiculata (Bangiophyceae, Rhodophyta) was carried out, by which we confirmed when the meiotic phase occurred in its life cycle. Moreover, the early development and morphogenesis of Py. suborbiculata blades were also observed. The red-type pigmentation mutant (HT) was used as the female parent, and the green-type (LT) was used as the male parent. The F1 gametophytic blades developed from conchospores which were released from heterozygous conchocelis showed two parental colors (G and R) and two new colors (W, wild-like color; Y, yellow-brown color). Linear segregation of colors occurred in the F1 blades, forming color-sectored blades with 2–4 sectors. Of the F1 gametophytic blades, 89.2% were color-sectored, and the proportion of two-sectored and three-sectored blades was the highest, accounting for 41.5% and 40%, respectively. Sectors of these four colors appearing in the sectored blades were in a ratio of 1054 R:1030 G:824 W:591 Y, which was approximately 1 R:0.98 G:0.78 W:0.56 Y. These results indicated that the meiosis of Py. suborbiculata occurred during the initial two-cell divisions of the germinating conchospores. Linear tetrad resulted from the meiosis continued to develop, the first vertical division started when the blades were divided into 6–9 cells, and the blade shape began to widen. Afterwards, a large amount of monospores was formed and released from the tip of blades, eventually resulting in round- or kidney-shaped blades in Py. suborbiculata.

Convergent evolution of cytochrome P450s underlies independent origins of keto-carotenoid pigmentation in animals.

Keto-carotenoids contribute to many important traits in animals, including vision and coloration. In a great number of animal species, keto-carotenoids are endogenously produced from carotenoids by carotenoid ketolases. Despite the ubiquity and functional importance of keto-carotenoids in animals, the underlying genetic architectures of their production have remained enigmatic. The body and eye colorations of spider mites (Arthropoda: Chelicerata) are determined by β-carotene and keto-carotenoid derivatives. Here, we focus on a carotenoid pigment mutant of the spider mite Tetranychus kanzawai that, as shown by chromatography, lost the ability to produce keto-carotenoids. We employed bulked segregant analysis and linked the causal locus to a single narrow genomic interval. The causal mutation was fine-mapped to a minimal candidate region that held only one complete gene, the cytochrome P450 monooxygenase CYP384A1, of the CYP3 clan. Using a number of genomic approaches, we revealed that an inactivating deletion in the fourth exon of CYP384A1 caused the aberrant pigmentation. Phylogenetic analysis indicated that CYP384A1 is orthologous across mite species of the ancient Trombidiformes order where carotenoids typify eye and body coloration, suggesting a deeply conserved function of CYP384A1 as a carotenoid ketolase. Previously, CYP2J19, a cytochrome P450 of the CYP2 clan, has been identified as a carotenoid ketolase in birds and turtles. Our study shows that selection for endogenous production of keto-carotenoids led to convergent evolution, whereby cytochrome P450s were independently co-opted in vertebrate and invertebrate animal lineages.

Pigment mutants of the green microalga Chlamydomonas reinhardtii: morphological properties and photosynthetic performance

Pigment mutants CC-124y-1, СС-124у-2, and СС-124р-2 of the green microalga Chlamydomonasreinhardtii Dangeard were obtained by means of UV-induced mutagenesis. Morphological propertiesand photosynthetic activity of these mutants were examined. The mutants displayed a lowered contentof chlorophyll b and carotenoids. Analysis of fluorescence induction curves revealed the decreased ratesof electron transport in photosystem II (PSII), the increase in the fraction of QB-nonreducing centers, andthe enhancement of nonphotochemical fluorescence quenching. The mutations had no direct impact onoxidation of pigment P700 in PSI and on the decline of delayed fluorescence. The parameters of induction curves of prompt and delayed fluorescence are proposed for use in early diagnostics of UV-inducedmutagenesis. The pigment mutants obtained can be applied in biomonitoring studies.Key words: Chlamydomonas reinhardtii, chlorophyll a fluorescence, delayed fluorescence, JIP test,photosynthesis.

Phenomenon of cell breakdown and phenotypic observation on surviving blades in inter-species hybridization progeny between pigment mutants of Pyropia haitanensis and P. acanthophora (Bangiales, Rhodophyta)

Pyropia haitanensis is a species of red algae native to China. Its meiosis occurs during the first two divisions of germinating conchospores and a tetrad with different genotypes is formed in the second division. Then each cell continues to undergo mitosis and finally, the conchosporeling develops into a genotype chimeric blade. Conchospores were formed from the inter-species hybridization between a red-type pigmentation mutant of P. haitanensis and a green-type pigmentation mutant of Pyropia acanthophora, among which, a phenomenon of serious cell breakdown occurred in the early developmental stage of germination. The phenomenon of cell breakdown occurred not only in the direct products of meiosis (conchosporelings containing 2–4 cells), but also in conchosporelings whose cell number was more than four, but large-scale cell breakdown occurred in conchosporelings containing 3–4 cells. After 35 days in culture, 99.2% of conchosporelings was unable to form thallus due to the heavy breaking down of somatic cells and only about 0.8% of the conchosporelings could form thallus, but most of them could not develop from the complete tetrads but just developed from 1 to 2 surviving tetrad cells. The F1 gametophytic blades developed from conchospores showed two parental colors (R and G) and four new colors (R’, redder in color than R; G’, lighter in color than G; T, tawny in color; W, light brown in color). The results confirmed that the hybrid breakdown of Pyropia could not only be used to assist in the more accurate identification of species; but also could be used to select new strains with recombination advantage from the surviving offspring.

An efficient method to generate xenograft tumor models of acute myeloid leukemia and hepatocellular carcinoma in adult zebrafish

Zebrafish is emerging as a promising model for the study of human cancers. Several xenograft models of zebrafish have been developed, particularly in larval stages (<48 h post fertilization) when the immune system of fish is not developed. However, xenografting in adult zebrafish requires laborious and transient methods of immune suppression (γ- irradiation or dexamethasone) that limits engraftment and survival of the tumor or fail to recapitulate specific characteristics of malignancies. Thus, the availability of a simple protocol to successfully engraft adult zebrafish, remains a challenge. The current study addresses this limitation and describes a robust method of xenografting in adult zebrafish. We describe a protocol that involves pre-conditioning of Casper, a pigmentation mutant of zebrafish with busulfan that led to a higher rate of engraftment of hepatocellular carcinoma and acute myeloid leukemia cells. To further ascertain the homing characteristics of the injected cancer cells, we transplanted adult zebrafish by two routes of administration and then studied their compartmentalization. This model presents a valuable alternative to rodents to study the biology of these cancers and also a cost-effective platform for evaluation of potential anti-cancer agents.

Unraveling the metabolic behavior in tomato high pigment mutants (hp-1, hp-2dg, ogc) and non ripening mutant (rin) during fruit ripening

A comprehensive research has been undertaken to explore the shift of primary metabolic flux with phenolic acid profile during transition of mature green to ripe stage of tomato(Solanum lycopersicum) high pigment (hp-1, hp-2dg and ogc) and non-ripening (rin) mutants. High pigment mutants revealed higher response for the amino acids like phenylalanine, tryptophane, proline, lysine, glutamicacid, serine, gamma aminobutyric acid, glutamine, aspartic acid, glycine, alanine, leucine and threonine compared to ‘Ailsa Craig’ along with the induced response in terms of organic acids viz. oxoglutaric acid, acetic acid, maleic acid, succinic acid,phosphoric acid, fumaric acid,ascorbic acid, tartaric acid, cis-aconitic acid, lactic acid, malic acid and methyl maleic acid. Cell wall sugars were lower in induced shelf-life mutant of hp-2dg and rin. Amongst phenolic acids chlorogenic acid, protocatechuic acid, caffeic acid, ellagic acid, gallic acid, 4-hydroxybenzoic acid showed increased response for high pigment mutants. Total phenol content was lower in high pigment mutants with no significant change for flavonoids. High pigment tomato mutants are metabolically explored for first time through Gas Chromatography Mass Spectrometry and Ultra Performance Liquid Chromatography. Fruit tissue specific primary metabolic shifts along with phenolic acids profile during ripening of tomato high pigment and non-ripening mutants has also been established for the first time. This metabolite kinetics may provide the foundation for utilizing these natural high pigment and non-ripening mutants for marker assisted breeding and metabolite engineering in future.

Genotype determination of the OPN1LW/OPN1MW genes: novel disease-causing mechanisms in Japanese patients with blue cone monochromacy

Blue cone monochromacy (BCM) is characterized by loss of function of both OPN1LW (the first) and OPN1MW (the downstream) genes on the X chromosome. The purpose of this study was to investigate the first and downstream genes in the OPN1LW/OPN1MW array in four unrelated Japanese males with BCM. In Case 1, only one gene was present. Abnormalities were found in the promoter, which had a mixed unique profile of first and downstream gene promoters and a −71A > C substitution. As the promoter was active in the reporter assay, the cause of BCM remains unclear. In Case 2, the same novel mutation, M273K, was present in exon 5 of both genes in a two-gene array. The mutant pigments showed no absorbance at any of the wavelengths tested, suggesting that the mutation causes pigment dysfunction. Case 3 had a large deletion including the locus control region and entire first gene. Case 4 also had a large deletion involving exons 2–6 of the first gene. As an intact LCR was present upstream and one apparently normal downstream gene was present, BCM in Case 4 was not ascribed solely to the deletion. The deletions in Cases 3 and 4 were considered to have been caused by non-homologous recombination.

De novo transcriptome analysis of an albino mutant Pasphiopedilum pacific shamrock reveals reduced expression of genes related to chloroplast biosynthesis and division

Paphiopedilum pacific shamrock is an orchid with high ornamental value. Understanding the mechanisms responsible for leaf color in albino mutants is important for ornamental development and breeding. In this study, we compared the leaf photosynthetic pigment content and transcriptome of albino mutants ppa01 and wild-type P. pacific shamrock. Photosynthetic pigment in mutants was less than 2% of the wild type and chl a/b was 60% less than the wild type. Transcriptome sequencing yielded 6.27 Gb and 5.67 Gb clean data from the mutant and wild-type leaves, respectively. De novo assembly yielded 104,763 unigenes with 15,400 greater than 1 kb in length. In unigene expression analysis, 3170 differentially expressed genes (DEGs) were identified with 2231 (70.38% of total DEGs) down-regulated. Results from GO and KEGG enrichment analysis, KEGG pathway analysis and qPCR suggest that the reduction of chloroplast biosynthesis and division in the mutant was due to low expression levels of ppGLK1 and ppFtsz. Mutants were associated with fewer chloroplasts in leaf cells, abnormal chloroplast structures, impaired chlorophyll biosynthesis, and thus reduced total chlorophyll and carotenoid contents. Furthermore, down-regulated expression of ppNYC1 reduced transformation of chlorophyll b into chlorophyll a, leading to a chl a/b decline. The research will guide future studies of leaf pigment mutations and the breeding of P. pacific shamrock.

Characterization of Porphyra lucasii pigment mutant induced by gamma irradiation

SUMMARYThe present study describes the isolation of a pigment mutant strain of Porphyra lucasii, which was obtained using gamma irradiation. Gametophytic blades of this seaweed were irradiated with gamma rays at doses of 500 and 1000 Gy. After irradiation, red color was observed in the blades exposed to the 500 Gy dose. Single somatic cells from these irradiated blades were isolated from archespores and then cultured to obtain pigment mutant blades that are referred to as 500GR in this investigation. Evaluation of genetic variations in these blades was performed using random amplification of polymorphic DNA (RAPD) analysis. Chlorophyll content, antioxidant activity, and total phenolic content were found to be higher in 500GR than in the wild type (WT) algal strain. Protein extracts obtained from the blades were examined using 2D gel electrophoresis; changes in the proteome were finally analyzed using MS/MS. A total of 216 proteins were detected. From these, nine proteins were over‐expressed in the pigment spots in 500GR as compared to WT. These results indicate that the over‐expressed proteins are either antioxidant enzymes or photosynthesis‐related proteins.

Pyomelanin produced by Vibrio cholerae confers resistance to predation by Acanthamoeba castellanii

Protozoan predation is one of the main environmental factors constraining bacterial growth in aquatic environments, and thus has led to the evolution of a number of defence mechanisms that protect bacteria from predation. These mechanisms may also function as virulence factors in infection of animal and human hosts. Whole transcriptome shotgun sequencing of Vibrio cholerae biofilms during predation by the amoebae, Acanthamoeba castellanii, revealed that 131 transcripts were significantly differentially regulated when compared to the non-grazed control. Differentially regulated transcripts included those involved in biosynthetic and metabolic pathways. The transcripts of genes involved in tyrosine metabolism were down-regulated in the grazed population, which indicates that the tyrosine metabolic regulon may have a role in the response of V. cholerae biofilms to A. castellanii predation. Homogentisate 1, 2-dioxygenase (HGA) is the main intermediate of the normal L-tyrosine catabolic pathway which is known to auto-oxidize, leading to the formation of the pigment, pyomelanin. Indeed, a pigmented mutant, disrupted in hmgA, was more resistant to amoebae predation than the wild type. Increased grazing resistance was correlated with increased production of pyomelanin and thus reactive oxygen species (ROS), suggesting that ROS production is a defensive mechanism used by bacterial biofilms against predation by amoebae A. castellanii.

Identification of YfiH and the Catalase CatA As Polyphenol Oxidases of Aeromonas media and CatA as a Regulator of Pigmentation by Its Peroxyl Radical Scavenging Capacity.

Pyomelanin is the major constituent of pigment in melanogenic Aeromonas strains of bacteria. However, eumelanin, synthesized from tyrosine via L-DOPA and polyphenol oxidases (PPOs), may also be present in this genus since L-DOPA is frequently detected in culture fluids of several species. To address this question, we used a deletion mutant of Aeromonas media strain WS, in which pyomelanin synthesis is completely blocked under normal culture conditions. When tyrosine was supplied to the medium, we observed residual melanin accumulation, which we interpret as evidence for existence of the DOPA-melanin pathway. We traced enzymatic activity in this bacterium using native-polyacrylamide gel electrophoresis. Two PPOs: YfiH, a laccase-like protein, and CatA, a catalase, were identified. However, neither protein was critical for the residual pigmentation in pyomelanin-deficient mutant. We speculate that eumelanin synthesis may require other unknown enzymes. Deletion of yfiH did not affect pigmentation in A. media strain WS, while deletion of the CatA-encoding gene katE resulted in a reduction of melanin accumulation, but it started 9 h earlier than in the wild-type. Since catalases regulate reactive oxygen species levels during melanogenesis, we speculated that CatA affects pigmentation through its peroxyl radical scavenging capacity. Consistent with this, expression of the catalases Hpi or Hpii from Escherichia coli in the katE deletion strain of A. media strain WS restored pigmentation to the wild-type level. Hpi and Hpii also exhibited PPO activity, suggesting that catalase may represent a new class of PPOs.

Transcriptome analysis reveals potential genes involved in flower pigmentation in a red-flowered mutant of white clover (Trifolium repens L.)

White clover (Trifolium repens L.) has been cultivated for ornamental use because of its flowers, leaf marks and creeping habit. Although a mutation in flower color is very infrequent in this species, the red-flowered mutant of white clover was a novel germplasm for ornamental white clover breeding. The mechanism of flower pigmentation in white clover is still limited because of the rarity of mutation materials and the lack of genomic data. In this study, two cDNA libraries from red-flowered white clover mutant between sunlight-exposed plants and shade-treated plants, respectively, were used for transcriptome sequencing. A total of 157,964 unigenes with an average length of 728bp and a median length of 1346bp were isolated. A large number of differentially expressed genes (6282) that were potentially involved in multiple biological and metabolic pathways, including anthocyanin flavonoid biosynthetic pathway and flavonoid biosynthetic pathway, were obtained, 70 of which could be identified as putative homologues of color-related genes. Furthermore, eight key candidate genes (CHS, F3′H, F3′5′H, UFGT, FLS, LAR, ANS, and DFR) in flavonoid biological synthesis pathway were identified by quantitative real-time PCR (qRT-PCR). Mass sequence data obtained by RNA-Seq of white clover and its red-flowered mutant provided basic sequence information and a platform for future molecular biological research on the red flower trait.

High pigments and atroviolacea double mutant of tomato (Solanum lycopersicum) with high pigments content

Tomato is known to contain nutritional components with several types of health-promoting actions. Lycopene which is an antioxidant present in tomatoes has been identified as being responsible for the beneficial effect of tomatoes. With the aim of increasing the antioxidants contents of tomato, two dark fruited single recessive mutants were crossed to produce double mutant with higher antioxidants contents. The high pigment (hp) mutant of tomato characterized by dark green fruits, when it is immature and deep red mature fruit which is a monogenic mutant of tomato, was crossed with atroviolacea (atv) monogenic non-allelic mutant which had the same phenotype. Although it was easy to distinguish the hpatv segregating double mutant in the f2 generation, the conformation of the hpatv double mutant was made genetically. The double mutant showed darker phenotype in stem, leaves,and much darker fruits due to higher pigments content. The double mutant qualitatively showed higher pigments content which is of great nutritional value the detailed estimation of the quantitative differences in pigments of the wild type, singlemutants and constructed mutant is recommended.

A defect in the mitochondrial protein Mpv17 underlies the transparent casper zebrafish

The casper strain of zebrafish is widely used in studies ranging from cancer to neuroscience. casper offers the advantage of relative transparency throughout adulthood, making it particularly useful for in vivo imaging by epifluorescence, confocal, and light sheet microscopy. casper was developed by selective breeding of two previously described recessive pigment mutants: 1) nacre, which harbors an inactivating mutation of the mitfa gene, rendering the fish devoid of pigmented melanocytes; and 2) roy orbison, a mutant with a so-far unidentified genetic cause that lacks reflective iridophores. To clarify the molecular nature of the roy orbison mutation, such that it can inform studies using casper, we undertook an effort to positionally clone the roy orbison mutation. We find that roy orbison is caused by an intronic defect in the gene mpv17, encoding an inner mitochondrial membrane protein that has been implicated in the human mitochondrial DNA depletion syndrome. The roy orbison mutation is phenotypically and molecularly remarkably similar to another zebrafish iridophore mutant called transparent. Using Cas9-induced crispants and germline mutants with a disrupted mpv17 open reading frame, we show in trans-heterozygote embryos that new frameshift alleles of mpv17, roy orbison, and transparent fail to complement each other. Our work provides genetic evidence that both roy orbison and transparent affect the mpv17 locus by a similar if not identical genetic lesion. Identification of mpv17 mutants will allow for further work probing the relationship between mitochondrial function and pigmentation, which has to date received little attention.